With Down Syndrome Decreased Naive T Cell Numbers in Children

Decreased Thymic Output Accounts for Decreased Naive T Cell Numbers in Children with Down Syndrome

This information is current as Beatrijs L. P. Bloemers, Louis Bont, Roel A. de Weger, of September 27, 2021. Sigrid A. Otto, Jose A. Borghans and Kiki Tesselaar J Immunol 2011; 186:4500-4507; Prepublished online 23 February 2011; doi: 10.4049/jimmunol.1001700

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Decreased Thymic Output Accounts for Decreased Naive T Cell Numbers in Children with Down Syndrome

Beatrijs L. P. Bloemers,* Louis Bont,* Roel A. de Weger,† Sigrid A. Otto,‡ Jose A. Borghans,‡ and Kiki Tesselaar‡

Children with Down syndrome (DS) have low numbers of naive T cells and abnormal thymus development and function. Because next to thymic production, peripheral proliferation greatly contributes to naive T cell generation in healthy children, we examined the cause of reduced naive T cell numbers in children with DS. Compared with aged matched controls, the total number of signal joint TCR excision circles (sjTREC) per ml blood was reduced in DS. Reduced frequencies and absolute numbers of protein tyrosine kinase 7-positive recent thymic emigrants, but similar levels of naive T cell apoptosis and Ag-driven activation in DS, suggested that reduced thymic output and not increased peripheral loss of naive T cells caused the reduced sjTREC numbers. We found no support for defective peripheral generation of naive T cells in DS. In DS the naive T cells responded to IL-7 and, based on Ki-67 expression, Downloaded from had similar proliferation rates as in healthy controls. sjTREC content per naive CD8+ T cells was not increased, but even decreased, pointing to increased survival or peripheral generation of naive T cells in DS. In conclusion, we show in this study that reduced thymic output, but not reduced peripheral generation nor increased loss of naive T cells, results in the low naive T cell numbers found in DS. The Journal of Immunology, 2011, 186: 4500–4507. http://www.jimmunol.org/ he most common chromosomal abnormality of live-born the thymus as seen in elderly. Although thymocyte maturation infants is Down syndrome (DS). Children with DS have appears disturbed, normal mature T cells expressing CD3 and T a high morbidity because of respiratory tract infections (1, TCRab are found in peripheral blood (21, 22). 2). In addition, various immunological impairments are associated Establishment and maintenance of the peripheral naive T cell with DS. Children with DS have a high incidence of hematologic compartment are dynamic processes. T cells are produced by the malignancies and autoimmune diseases like hypothyroidism, ce- thymus and are released in peripheral blood as recent thymic liac disease, and diabetes mellitus (3–7). emigrants (RTE). Because these cells are the most proximal to the Studies on the adaptive immune system of DS have shown that thymus, they are essential for maintaining a diverse ab TCR total lymphocyte numbers are decreased in this population (8–14). repertoire. This proximity is also reflected by their high content of by guest on September 27, 2021 This holds true for the CD4+ and CD8+ T cell subsets, especially signal joint TCR gene excision circles (sjTRECS), which are in the first 2 y of life, but becomes less pronounced with in- circular DNA products of intrathymic V(D)J recombination. Pe- creasing age and eventually normalizes around the age of 16 y. ripheral proliferation of naive T cells and longevity are the other Subdivision of T cells into naive and memory cells showed that in mechanisms contributing to establishment and maintenance of the fact only the naive subsets are smaller compared with healthy naive T cell pool. Main drivers of these processes are IL-7 (23) controls (8, 9, 11–13). In contrast, the fraction of CD8+ T cells and TCR-MHC/self-peptide ligand interactions (24, 25). Based on within the T cell compartment is increased, and the fraction of phenotypic markers, several naive CD4+ T cell subsets with dif- CD4+ T cells decreased (9). Percentages of naive cells have been ferent dynamic histories can be distinguished (24). Recently, described to be decreased and memory and effector subsets to be protein tyrosine kinase 7 (PTK7) has been described as a novel increased in both CD4+ and CD8+ T cells (15–17). Several studies marker for CD4+ RTE (26). Naive CD31-positive and PTK7- have ascribed the immunologic impairment of T cells in DS to positive CD4+ T cells are considered to be the subset most prox- abnormal thymus development and function (16, 18–21). Children imal to the thymus, and MHC/self-peptide ligand-induced pro- with DS show increased cortical thymocyte depletion, cystic liferation of these cells has been suggested to lead to loss of PTK7 changes, and fibrosis, suggestive of an accelerated involution of and subsequently CD31 expression. IL-7 preferentially induces proliferation of the CD31-positive subset, thereby downmodu- lating CD127 expression, but not inducing any other phenotypical *Department of Paediatric Infectious Diseases and Immunology, University Medical changes (27). † Centre Utrecht, Utrecht, The Netherlands; Department of Pathology, University Roat et al. (17, 28) showed that children with DS have lower Medical Centre Utrecht, Utrecht, The Netherlands; and ‡Department of Immunology, University Medical Centre Utrecht, Utrecht, The Netherlands percentages of sjTREC-positive cells in PBMCs compared with Received for publication May 21, 2010. Accepted for publication January 17, 2011. controls. They suggested that this was the result of thymic impairment. Although this conclusion could explain their observa- Address correspondence and reprint requests to Dr. Louis Bont, Department of Paediatric Infectious Diseases and Immunology, University Medical Centre Utrecht, tion, sjTREC dynamics and T cell homeostasis are also influenced P.O. Box 85090, 3508 AB Utrecht, The Netherlands. E-mail address: L.J.Bont@ by cellular life span and division (29). In the current study, we umcutrecht.nl addressed naive T cell dynamics in children with DS now in- The online version of this article contains supplemental material. corporating thymic output, T cell proliferation, and T cell loss by Abbreviations used in this article: DS, Down syndrome; PTK7, protein tyrosine Ag-driven differentiation or apoptosis. From our combined results, kinase 7; RTE, recent thymic emigrant; sjTREC, signal joint TCR excision circle. we conclude that decreased thymic output is responsible for the Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 low naive T cell numbers in children with DS. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1001700 The Journal of Immunology 4501

Materials and Methods dition, in a limited number of patients, expression of CD25, CD31, CD38, Study population HLA-DR, CD127, Ki67, and PTK7 within naive and memory subsets was determined. Examples of the gating strategy used are shown in Supple- Whole blood samples were obtained by venapuncture from healthy children mental Fig. 1. Absolute lymphocyte count was determined using patented with DS and controls. Forty-seven children with DS who attended the Multi Angle Polarized Scatter Separation plus three-color fluorescent on outpatient clinic of the Wilhelmina Children’s Hospital Utrecht, The a CELL-DYN Sapphire (Abbott Diagnostics) and was used to calculate Netherlands, were included in six age groups from 0.1 to 12 y. Because the absolute numbers of the indicated lymphocyte subsets by multiplying the largest developmental changes in the immune system are described in percentage of the subset as obtained by flow cytometry with the ab- approximately the first 2 y of life, three times more children were included solute lymphocyte number. in the age group of 0–2 y (n = 16) compared with the other age groups, in which five children per 2-y stratum were included (30, 31). Children with sjTREC analysis DS with a history of surgery in the previous 2 mo were excluded. An equal sjTREC numbers were determined by real-time PCR on genomic DNA of number of age-matched, otherwise healthy children, who were admitted to CD4+ and CD8+ T cells, as described previously (29, 33). DNA was pu- the hospital for elective urologic, plastic, ophthalmologic, or general sur- rified using the NucleoSpin Blood QuickPure, according to manufacturer’s gery, were included as controls. To minimize interference on immunologic instructions (Machery-Nagel). sjTREC content per CD4+ or CD8+ T cell parameters, blood was drawn prior to or directly after anesthesia was was calculated by dividing the sjTREC content/mg DNA by 150,000 (as- given. None of the children showed signs of acute infection at the time that suming that 1 mg DNA corresponds with 150,000 T cells). Total numbers blood was drawn. Five children with DS and eight control children used of sjTRECs were calculated as the sjTREC content per CD4+ or CD8+ prophylactic antibiotics at the time of sampling. Four to 10 ml blood was T cell multiplied by the absolute CD4+ or CD8+ T cell count/ml. drawn from each individual. In four children with DS and three controls, the absolute lymphocyte count could not be determined. PBMCs could not IL-7 ELISA be isolated at all in three children of both groups. FACS staining could not be performed in four children with DS and six healthy controls and only Plasma samples were frozen 3–12 h after they were obtained. A short t1/2 of Downloaded from partially in the remaining children. For all cases parental written informed IL-7 might influence the results of our tests using samples with a wide time consent was obtained. The research protocol was approved by the medical frame before freezing. To exclude time as a possible dependent factor in ethics committee of the University Medical Centre Utrecht. the results of our test, we examined the influence of time until freezing of the sample on IL-7 plasma levels in three healthy donors. No significant Cell preparation and cultures differences were found in IL-7 plasma levels of samples frozen between 3 and 12 h after sampling (data not shown). None of the samples had been PBMCs were isolated from heparinized blood samples, and plasma was thawed previously. After thawing, samples were analyzed using an ELISA isolated from EDTA-anticoagulated blood samples. PBMCs were obtained (Quantikine HS; R&D Systems), according to the manufacturer’s recom- http://www.jimmunol.org/ by Ficoll-Paque density-gradient centrifugation and stored in liquid ni- mendations. All samples were run in duplicate. A standard curve was pre- trogen until further processing. CD4+ and CD8+ T cells were purified from pared by serial dilutions. thawed PBMCs by magnetic bead separation using the MiniMACS mul- tisort kit, according to manufacturer’s instructions (Miltenyi Biotec). To Statistical analysis measure sjTREC content within naive (CD27+CD45RO2) and memory + + + + Differences in results between children with DS and healthy controls were (CD27 CD45RO ) CD4 and CD8 T cells, these subsets were isolated by compared using x2 test, Student t test, or nonparametric tests in case of cell sorting on a FACSAria (BD Biosciences). PBMCs were cultured with unequal distribution between the groups. Values are expressed as the or without IL-7 at a final concentration of 10 ng/ml IL-7 (Sigma-Aldrich) means 6 SEM. In all analysis, regression analysis, including sex and age, in RPMI 1640/10% FCS culture medium for 7 d. For the apoptosis assay, was performed to evaluate whether differences existed between children

cryopreserved PBMCs were thawed and cells were either stained directly by guest on September 27, 2021 with DS and healthy controls. All statistical analyses were performed using with the apoptotic marker annexin V (BD Biosciences) or after overnight the software program SPSS for Windows (version 12.0.2; SPSS, Chicago, culture in medium. IL). A p value #0.05 was considered significant. A sensitivity analysis was Flow cytometry performed to determine whether a history of corrective surgery for congenital heart disease in children with DS could have influenced the out- Thawed cryopreserved PBMCs were used for characterization of the come of the results of this study. All analyses were repeated for children T cell compartment. PBMCs were stained with mAbs to CD3-Pacific Blue, without corrective heart surgery. CD4-Pacific Blue, CD4-PerCP-Cy5.5, CD4-allophycocyanin-Cy7, CD8- PerCP-Cy5.5, CD8-allophycocyanin-Cy7, CD8-Amcyan, CD25-PE, CD27- allophycocyanin, CD27-allophycocyanin-Cy7, CD31-PE, CD38-PerCP- Results Cy5.5, CD45RO-PE-Cy7, CD127-PE, HLA-DR-FITC, and HLA-DR-PE Forty-seven children with DS, ages 0–12 y, were included in the (BD Biosciences). Intracellular staining was performed to measure expres- study. An equal number of healthy age-matched children admitted sion of Ki67 (DakoCytomation). In short, PBMCs were stained, fixed, and to the hospital for elective surgery were included as controls. permeabilized with Cytofix/Cytoperm (BD Biosciences) and washed twice with permeabilization wash buffer. Next, cells were stained with Ab directed Children were divided in eight age strata of 2 y with five to six against Ki-67, washed twice with permeabilization buffer, and resuspended children each. We used narrower strata in the first 2 y of life be- in FACS buffer. Affinity-purified rabbit IgG anti-mouse PTK7 was provided cause the largest immunological changes are found during those by X. Lu (Department of Cell Biology, University of Virginia, Charlottes- years. Because of restrictions in blood volume that could be drawn ville, VA).For surface PTK7 staining, Alexa Fluor 488–conjugated goat anti- and concomitant yield of PBMCs, some experiments could not rabbit IgG was used as a secondary Ab. As a control for aspecific binding, cells were stained with an identical mixture without the anti-mouse PTK7 be performed in all 47 children (for details, see Materials and Ab. Annexin V staining was used according to manufacturer’s protocol to Methods). In our study group of children with DS, 20 children determine the fraction of apoptotic cells. In short, after surface staining of with DS had congenital heart disease in some degree. Six of them T cell subsets, cells were washed, incubated with annexin VAbs for 15 min, had no corrective surgery. In four children, only analysis of IL-7 and washed again. Cells were resuspended in annexin V buffer and analyzed. Forward/Sideward scatter was used to exclude dead cells. Cellular fluores- levels has been performed. Ten of 14 children, who had corrective cence was measured using a LSRII flow cytometer (BD Biosciences) and heart surgery, had an age .6.5 y when blood was drawn. analyzed with FACS Diva software (BD Biosciences). For each sample, + + minimally 50,000 events were present in the lymphocyte gate (see Supple- Decreased absolute numbers of naive CD4 and CD8 T cells mental Fig. 1 for gate definition). FACS results were only included in the We confirmed that children with DS have lower absolute numbers . analysis if for a specific subpopulation 25 events were counted. of naive CD4+ and CD8+ T cells compared with healthy controls 9 + Population definitions (0.91 versus 1.84 3 10 /l naive CD4 , p , 0.001 and 0.41 versus 0.83 3 109/l naive CD8+, p , 0.001) (Fig. 1) (8–13). This effect CD8+CD3+ T cells were subdivided into naive (CD27+CD45RO2), memory (CD27+CD45RO+), memory effector (CD272CD45RO+), and effector was seen at all ages, although differences between groups became (CD272CD45RO2) subsets (32). In analogy, we have used the same smaller with increasing age. Absolute numbers of naive T cells definition of naive and Ag-experienced subsets for CD4+ T cells. In ad- were negatively correlated with age in both groups. Normal ab- 4502 T CELL DYNAMICS IN CHILDREN WITH DOWN SYNDROME

FIGURE 1. Children with DS have decreased absolute naive T cell counts. Age distribution of absolute numbers of naive CD4+ (A) and naive CD8+ (B)T cell subsets in children with DS (n = 36) compared with healthy controls (HC) (n = 35). Statistical analysis is shown for the difference between the group of children with DS and the group of healthy controls. Regression analysis showed independent effects of DS and age. Downloaded from solute numbers of memory and effector subsets of CD4+ and versus 0.13 3 109/l, p = 0.001, respectively; Fig. 2A,2B). Re- CD8+ T cells were found in children with DS compared with gression analysis of total sjTREC numbers in CD4+ and CD8+ controls (Supplemental Fig. 2A,2B). T cells showed independent effects of DS and age. Next, we measured the absolute number and percentage of CD4+ RTEs as determined by the expression of PTK7 on naive CD4+ Decreased sjTRECs and RTE numbers in children with DS

T cells. In children with DS, the absolute number and the per- http://www.jimmunol.org/ Several studies have shown thymic abnormalities in children with centage of PTK7-positive cells within naive CD4+ T cells were DS. To assess the cause of low absolute numbers of naive T cells in decreased (0.10 versus 0.32 3 109/l, p , 0.001 and 10.2 versus children with DS, we ﬁrst measured total sjTREC numbers/ml 18.6%, p = 0.008, respectively; Fig. 2C,2D). Regression analysis blood. Both for CD4+ and CD8+ T cells we found total sjTREC of both absolute numbers and percentages of PTK7-positive cells numbers to be .2-fold decreased in children with DS compared within naive CD4+ T cells showed independent effects of DS and with controls (mean 0.23 versus 0.50 3 109/l, p = 0.003 and 0.07 age. by guest on September 27, 2021

FIGURE 2. Children with DS have lower TREC numbers and RTE numbers and frequency compared with healthy controls (HC). Age distribution of CD4+ (A) and CD8+ (B) TREC counts/l blood, and absolute numbers (C) and percentages (D) of PTK7+ RTE within naive CD4+ T cells in children with DS (n = 30, n = 30, n = 24, and n = 27, respectively) compared with healthy controls (n = 32, n = 32, n = 23, and n = 26, respectively). Statistical analysis is shown for the difference between the group of children with DS and the group of healthy controls. Regression analysis showed independent effects of DS and age. The Journal of Immunology 4503

No increased loss of peripheral naive T cells in DS possible disturbances in peripheral proliferation using the proliferation marker Ki67. Normal absolute numbers (Supplemental To determine whether reduced thymic output or increased loss + causes the observed reduction in sjTREC numbers and RTE, we Fig. 4A,4B) and percentages of naive CD4 T cells and naive CD8+ T cells expressing Ki67 were found in children with DS measured the level of peripheral naive T cell loss by flow + cytometry. We analyzed the percentage of naive CD4+ and CD8+ compared with controls (Fig. 4A,4B). For naive CD4 T cells, T cells expressing the apoptosis marker annexin V. Annexin ex- CD31 is a thymic proximity marker, and loss of CD31 expression pression was analyzed ex vivo and after 16 h of culture in the has been suggested to reflect self Ag-driven proliferation (24, 25, 27). In line with the Ki-67 data, analysis of the fraction of cells not presence or absence of polyclonal stimulation with anti-CD3 + mAb. Under all circumstances there was no difference between expressing CD31 among naive CD4 T cells was similar com- the fraction of apoptotic naive CD4+ and CD8+ T cells between pared with healthy controls (33.3 versus 28.7%, p = 0.09; Fig. 4C). children with DS and healthy controls (Fig. 3A,3B). Thus, both analyses showed no evidence for reduced peripheral Next, we analyzed the level of Ag-induced activation. We proliferation as a cause for the reduced naive T cell numbers in reasoned that, as seen in HIV infection, increased Ag-induced DS. activation could lead to naive T cell loss. The children with DS No indication for defective IL-7–dependent peripheral in this study did not show any clinical signs of acute or chronic proliferation or survival infection. We used expression of CD25 in naive T cells as a measure of recent antigenic activation of CD8+ T cells (34) (our IL-7 is a cytokine important for T cell survival and proliferation, personal observation). No differences were found in the absolute especially of naive T cells (23). Its production is regulated by an number of recently activated (CD25+CD45R02CD27+)CD8+ IL-7R–mediated feedback loop, which together with the utiliza- Downloaded from T cells (Fig. 3C). Analysis of T cell activation using the activation tion by T cells determines the serum and tissue levels of the cy- markers CD38+ and HLA-DR+ gave also no indications for in- tokine. In human lymphopenic settings, IL-7 plasma levels are creased T cell activation, as the absolute numbers of CD38+HLA- increased and are thought to increase low-affinity TCR-induced DR+ CD4+ and CD8+ T cells were similar in DS children compared proliferation and enhance survival of naive T cells (38–40). We with healthy controls (Supplemental Fig. 3A,3B). Because we investigated whether IL-7 influenced peripheral naive T cell found no indications of increased peripheral naive CD4+ or CD8+ numbers in DS. In children with DS, significantly higher IL-7 http://www.jimmunol.org/ T cell loss, we concluded that in children with DS the reduction in plasma levels were found, which was not affected by age (7.5 sjTREC numbers and RTE resulted from decreased thymic output. versus 5.8 pg/ml, p = 0.03; Fig. 5A). However, no correlation between IL-7 plasma level and number of total (Supplemental Fig. No indication for reduced peripheral generation of naive 5A,5B) and naive CD4+ or CD8+ T cells was found (Supplemental T cells Fig. 5C,5D). In vitro stimulation with IL-7 showed a normal Besides thymic output, peripheral proliferation contributes largely capacity to respond to IL-7 in children with DS, as down- to the establishment of the naive T cell pool (35–37). We assessed modulation of CD127, the limiting component of the IL-7R, was by guest on September 27, 2021

FIGURE 3. Children with DS have normal peripheral loss of naive CD4+ and CD8+ T cells. The fraction of annexin V+ naive CD4+ (A) and CD8+ (B) T cells was used as a marker for apoptosis by ﬂow cytometry in children with DS (n = 24 and n = 23, respectively) and healthy controls (HC) (n = 28 and n = 26, respectively). The absolute numbers of CD25+ naive CD8+ (C) T cells were determined as a measure for Ag-induced activation by ﬂow cytometry in children with DS (n = 28) and healthy controls (n = 21). 4504 T CELL DYNAMICS IN CHILDREN WITH DOWN SYNDROME

FIGURE 4. Children with DS show normal peripheral generation of naive CD4+ and CD8+ T cells. Flow cytometric analysis of relative numbers of Ki67+ naive CD4+ (A) and CD8+ (B) T cells in children with DS (n = 37) and healthy controls (HC) (n = 37). Flow cytometric analysis of relative numbers of CD312 naive CD4+ (C) T cells in children with DS (n = 40) and healthy controls (n = 37). Downloaded from http://www.jimmunol.org/

similar (data not shown). Ex vivo analysis of CD127 expression our hypothesis and even suggests increased proliferation or de- on naive T cells showed small increases in relative fraction of creased loss in children with DS. by guest on September 27, 2021 CD1272 within naive CD4+ (Fig. 5B) and CD8+ (Fig. 5C) subsets We performed a sensitivity analysis in which all analyses of this (24.6 versus 18.9%, p = 0.05 and 23.6 versus 12.0%, p , 0.001, study were repeated, excluding children with a history of corrective respectively), suggestive of increased per cell IL-7 utilization and heart surgery, to analyze the possible contribution of perioperative increased survival or proliferation. Regression analysis showed thymectomy in those children. All conclusions remained identical independent effects of age and DS in the CD4 subset and no effect without loss of statistical significance for any analysis. of age in the CD8 subset. Thus, despite increased IL-7 plasma levels, we found no indications for a role of IL-7 in decreasing Discussion peripheral proliferation in children with DS. Histopathologic studies of thymic tissue in DS have shown increased involution of the thymus and altered patterns of maturation Cumulative effects over time: sjTREC content analysis of thymocytes (19, 21, 22). In addition, several studies have de- To corroborate our conclusion that reduction in peripheral pro- scribed low numbers of T cells, decreased cytokine production liferation and increase of naive T cell loss are not the cause of upon antigenic stimulation, and diminished Ab-dependent T cell- reduced naive T cell numbers in children with DS, we measured mediated cytotoxicity in DS (10, 13, 41). It was suggested that sjTREC content of the CD4+ and CD8+ T cell compartment thymic insufficiency causes peripheral T cell dysfunction in DS (Supplemental Fig. 6). In contrast to sjTREC numbers/ml blood, and that decreased thymic output is a causative factor for the low which reflect the balance between thymic production and T cell numbers of naive T cells in these children. sjTREC analysis is the loss, sjTREC content per CD4+ or CD8+ T cell reflects the balance most applicable tool to determine thymic output in humans, but between thymic output, T cell proliferation, and loss, and small the results should be interpreted with care (29). Reduced sjTREC changes in proliferation or survival rates will accumulate over contents might be indicative of reduced thymic output, but are also time in larger differences in the sjTREC content. When peripheral influenced by peripheral mechanism of T cell maintenance. Be- proliferation or survival per cell is decreased, the sjTREC content cause sjTRECs are circles of DNA spliced off during TCR rear- per T cell will become relatively higher. In agreement with our rangement that do not replicate during mitosis, they are diluted hypothesis, no significant changes were found in the sjTREC upon cell division. In addition, longevity of naive T cells influ- content of sorted naive CD4+ T cells in children with DS com- ences sjTREC content of cells by decreasing sjTREC content pared with healthy controls (median 0.15 versus 0.31, p = 0.08; when naive T cells tend to live longer. For this reason, absolute Fig. 6A). For sorted naive CD8+ T cells (Fig. 6B), decreased numbers of sjTRECs per milliliter blood are used to provide in- sjTREC content was found in children with DS compared with formation on thymic output, whereas the average sjTREC content healthy controls (median 0.17 versus 0.28, p = 0.04). Regression per cell is used for determination of the replicative history. The analysis showed independent effects of age and DS. The decreased combined results of our detailed study of T cell dynamics now put sjTREC content in sorted naive CD8+ T cells is also in line with forward that in children with DS decreased thymic output is solely The Journal of Immunology 4505 Downloaded from http://www.jimmunol.org/

FIGURE 5. Children with DS have normal IL-7–dependent peripheral proliferation and survival. Plasma levels of IL-7 (A) measured by ELISA in children with DS (n = 29) compared with healthy controls (HC) (n = 35) at different ages. Regression analysis showed no effect of age. Flow cytometric analysis of relative numbers of CD1272 naive CD4+ (B) and CD8+ (C) T cells within the respective naive T cell pools in children with DS (n = 36 and n = 30, respectively) and healthy controls (n = 37 and n = 34, respectively). Statistical analysis is shown for the difference between the group of children with DS and the group of healthy controls. Regression analysis showed independent effects of age and DS in the CD4+ subset and no effect of age in the CD8+ subset. responsible for the decreased naive T cell numbers, and that pe- To our knowledge, we are the first to report on RTEs identified by guest on September 27, 2021 ripheral mechanisms of naive T cell maintenance are fully func- by PTK7 in a large pediatric population after its description by tional in children with DS. In fact, based on the reduced sjTREC Haines et al. (26) Within this healthy pediatric cohort, percentages content in naive CD8+ T cells, we conclude that peripheral of RTE were similar to percentages described by Haines. Re- mechanisms counteract the reduced thymic output. Future studies gression analysis showed an age-dependent decline in the absolute on thymopoiesis, including analysis of thymic tissue, in particular numbers, but not frequency of PTK7-positive cells within naive of T cell subsets, their reactivity to IL-7, intrathymic precursor CD4+ T cells in the blood between the age of 0 and 13 y. This age- T cell proliferation, and the implication of growth hormones in independent frequency of RTE implies that no RTE-specific turn- thymopoiesis, might clarify the cause of decreased thymic output over changes take place with age, and therefore, that RTE and in children with DS. resident naive T cells are mostly regulated by the same mechanisms.

FIGURE 6. Children with DS have normal or even decreased TREC content compared with healthy controls (HC). To determine whether lower TREC counts in CD4+ and CD8+ T cells reﬂect lower fraction of naive T cells within children with DS, TREC contents of puriﬁed naive CD4+ (A) and CD8+ (B) T cells were measured in children with DS (n = 9) and healthy controls (n = 9). Statistical analysis is shown for the difference between the group of children with DS and the group of healthy controls. Regression analysis showed independent effects of age and DS. 4506 T CELL DYNAMICS IN CHILDREN WITH DOWN SYNDROME

In previous DS literature, accelerated thymic involution and In conclusion, to our knowledge, this is the first study that clinical findings, such as occurrence of Alzheimer’s disease at the formally shows decreased thymic output as the cause of the di- young age of 40–50 y, have been ascribed to early senescence. minished naive T cell pool in children with DS, confirming the Ageing of the immune system is difficult to define, but decline in hypothesis of thymic insufficiency. Evidence of an intrinsic naive naive T cell numbers, oligoclonal expansion of memory T cells T cell defect contributing to a diminished naive T cell pool in starting around the sixth decade of life, and loss of TCR diversity these children could not be provided. Our data suggested that IL- are important aspects (42). Although the decreased numbers of 7–driven peripheral mechanisms might counteract the reduced naive T cells support the concept of accelerated ageing, other thymic output. Future functional studies are needed to determine results are in apparent contrast. First, in accelerated ageing one whether increased peripheral proliferation and reduced TCR di- would expect that absolute naive T cell numbers and total sjTREC versity of naive T cells have a role in the morbidity described in counts are lower and keep declining over age at a higher rate than DS. in controls. In children with DS, a higher rate of decline was only found in the first 2–4 y. Secondly, with ageing, naive and memory CD8+ T cells are lost, whereas CD8+ effector T cells increase (43, Acknowledgments 44). In children with DS, over age no significant changes in ab- We thank P. van der Weide (Department of Pathology, University Medical solute or relative numbers of CD8+ memory and effector T cells Centre Utrecht, The Netherlands) for excellent technical assistance, F. Mie- dema (Department of Immunology, University Medical Centre Utrecht, The were shown. A third aspect of ageing is loss of TCR diversity + Netherlands) for useful comments on the research and manuscript, and caused by oligoclonal expansion, mostly within memory CD8 X. Lu (Department of Cell Biology, University of Virginia, Charlottesville, b Downloaded from T cells. When we used V spectratyping of PBMCs to compare VA) for providing the affinity-purified rabbit IgG anti-mouse PTK7. oligoclonality (45, 46) of children with DS and healthy controls, a larger number of TCR Vb families that were oligoclonal were seen in children with DS (our unpublished results). Because TCR Disclosures diversity was not measured within CD4+ and CD8+ T cell subsets, The authors have no financial conflicts of interest. it is unclear whether this loss of TCR diversity found in children with DS reflects a relative increase of memory T cells or clonal References http://www.jimmunol.org/ expansion associated with homeostatic maintenance of the naive 1. Bloemers, B. L., A. M. van Furth, M. E. Weijerman, R. J. Gemke, C. J. Broers, T cell compartment or a combination of both. If loss of TCR di- K. van den Ende, J. L. Kimpen, J. L. Strengers, and L. J. Bont. 2007. Down versity in children with DS is not explained by early senescence, it syndrome: a novel risk factor for respiratory syncytial virus bronchiolitis— a prospective birth-cohort study. Pediatrics 120: e1076–e1081. might still play a role in the higher susceptibility to infections in 2. Hilton, J. M., D. A. Fitzgerald, and D. M. Cooper. 1999. Respiratory morbidity children with DS. Future studies are needed to explore TCR di- of hospitalized children with Trisomy 21. J. Paediatr. Child Health 35: 383–386. 3. Goldacre, M. J., C. J. Wotton, V. Seagroatt, and D. Yeates. 2004. Cancers and versity and functional loss of the immune system at different ages immune related diseases associated with Down’s syndrome: a record linkage to further clarify the concept of ageing in patients with DS. Future study. Arch. Dis. Child. 89: 1014–1017. longitudinal studies that correlate these immunologic parameters 4. Scholl, T., Z. Stein, and H. Hansen. 1982. Leukemia and other cancers, anom- alies and infections as causes of death in Down’s syndrome in the United States by guest on September 27, 2021 to clinical data could give better insight in the role of decreased during 1976. Dev. Med. Child Neurol. 24: 817–829. naive T cells in the morbidity found in children with DS. 5. Selikowitz, M. 1992. Health problems and health checks in school-aged children The natural variance in values of the dynamic parameters, even with Down syndrome. J. Paediatr. Child Health 28: 383–386. 6. Turner, S., P. Sloper, C. Cunningham, and C. Knussen. 1990. Health problems in in a healthy population, can be quite substantial (∼4-fold, up to 10- children with Down’s syndrome. Child Care Health Dev. 16: 83–97. fold for TREC content). Next to genetic disposition, factors like 7. Yang, Q., S. A. Rasmussen, and J. M. Friedman. 2002. Mortality associated with Down’s syndrome in the USA from 1983 to 1997: a population-based study. age and infection status are known effectors. Recent or chronic Lancet 359: 1019–1025. infections were exclusion criteria for our study, to prevent the 8. Cocchi, G., M. Mastrocola, M. Capelli, A. Bastelli, F. Vitali, and L. Corvaglia. effect of infection status. Despite our relative small group sizes 2007. Immunological patterns in young children with Down syndrome: is there a temporal trend? Acta Paediatr. 96: 1479–1482. and the large variance, we were able to find significant age effects 9. Cossarizza, A., D. Monti, G. Montagnani, C. Ortolani, M. Masi, M. Zannotti, and differences between DS and healthy controls. More important, and C. Franceschi. 1990. Precocious aging of the immune system in Down for all results that were significantly different between DS and syndrome: alteration of B lymphocytes, T-lymphocyte subsets, and cells with natural killer markers. Am. J. Med. Genet. Suppl. 7: 213–218. healthy controls, regression analysis showed an effect of DS that 10. de Hingh, Y. C., P. W. van der Vossen, E. F. Gemen, A. B. Mulder, W. C. Hop, was independent of age. Based on the difference between DS and F. Brus, and E. de Vries. 2005. Intrinsic abnormalities of lymphocyte counts in children with Down syndrome. J. Pediatr. 147: 744–747. healthy controls, we concluded that thymic output and not dis- 11. Guazzarotti, L., D. Trabattoni, E. Castelletti, B. Boldrighini, L. Piacentini, turbed peripheral generation is responsible for the decreased P. Duca, S. Beretta, M. Pacei, C. Caprio, A. Vigan Ago, et al. 2009. number of naive T cells in children with DS. In line with this T lymphocyte maturation is impaired in healthy young individuals carrying trisomy 21 (Down syndrome). Am. J. Intellect. Dev. Disabil. 114: 100–109. conclusion, none of our analyses suggested intrinsic defects in cell 12. Kusters, M. A., E. F. Gemen, R. H. Verstegen, P. C. Wever, and E. De Vries. death or proliferation or increased T cell activation that would 2010. Both normal memory counts and decreased naive cells favor intrinsic explain the reduced T cell numbers in DS. In contrast, in DS defect over early senescence of Down syndrome T lymphocytes. Pediatr. Res. + 67: 557–562. trends toward decreased numbers of CD25 and percentages of 13. Lockitch, G., V. K. Singh, M. L. Puterman, W. J. Godolphin, S. Sheps, annexin V+ naive T cells and increased frequencies of Ki67+ and A. J. Tingle, F. Wong, and G. Quigley. 1987. Age-related changes in humoral and 2 cell-mediated immunity in Down syndrome children living at home. Pediatr. CD31 naive T cells were found, which would suggest compen- Res. 22: 536–540. satory peripheral mechanisms for the reduced thymic output. In 14. Verstegen, R. H., M. A. Kusters, E. F. Gemen, and E. De Vries. 2010. Down addition to age and infection status, corrective surgery for con- syndrome B-lymphocyte subpopulations, intrinsic defect or decreased T- lymphocyte help. Pediatr. Res. 67: 563–569. genital heart disease with the risk of thymectomy might have 15. Barrena, M. J., P. Echaniz, C. Garcia-Serrano, and E. Cuadrado. 1993. Imbalance influenced the results of our study. Fourteen of 47 children with of the CD4+ subpopulations expressing CD45RA and CD29 antigens in the DS have had corrective surgery because of significant congenital peripheral blood of adults and children with Down syndrome. Scand. J. Immu- nol. 38: 323–326. heart disease. In four of them, only analysis of IL-7 levels was 16. Murphy, M., and L. B. Epstein. 1992. Down syndrome (DS) peripheral blood performed. A sensitivity analysis comparing children with DS contains phenotypically mature CD3+TCR alpha, beta+ cells but abnormal proportions of TCR alpha, beta+, TCR gamma, delta+, and CD4+ CD45RA+ without corrective heart surgery and healthy controls confirmed cells: evidence for an inefficient release of mature T cells by the DS thymus. the conclusions from all analyses. Clin. Immunol. Immunopathol. 62: 245–251. The Journal of Immunology 4507

17. Roat, E., N. Prada, E. Lugli, M. Nasi, R. Ferraresi, L. Troiano, C. Giovenzana, 32. Hamann, D., P. A. Baars, M. H. Rep, B. Hooibrink, S. R. Kerkhof-Garde, M. Pinti, O. Biagioni, M. Mariotti, et al. 2008. Homeostatic cytokines and ex- M. R. Klein, and R. A. van Lier. 1997. Phenotypic and functional separation of pansion of regulatory T cells accompany thymic impairment in children with memory and effector human CD8+ T cells. J. Exp. Med. 186: 1407–1418. Down syndrome. Rejuvenation Res. 11: 573–583. 33. Pongers-Willemse, M. J., O. J. Verhagen, G. J. Tibbe, A. J. Wijkhuijs, V. de 18. Larocca, L. M., M. Piantelli, S. Valitutti, F. Castellino, N. Maggiano, and Haas, E. Roovers, C. E. van der Schoot, and J. J. van Dongen. 1998. Real-time P. Musiani. 1988. Alterations in thymocyte subpopulations in Down’s syndrome quantitative PCR for the detection of minimal residual disease in acute lym- (trisomy 21). Clin. Immunol. Immunopathol. 49: 175–186. phoblastic leukemia using junctional region specific TaqMan probes. Leukemia 19. Larocca, L. M., L. Lauriola, F. O. Ranelletti, M. Piantelli, N. Maggiano, 12: 2006–2014. R. Ricci, and A. Capelli. 1990. Morphological and immunohistochemical study 34. Uchiyama, T., S. Broder, and T. A. Waldmann. 1981. A monoclonal antibody of Down syndrome thymus. Am. J. Med. Genet. Suppl. 7: 225–230. (anti-Tac) reactive with activated and functionally mature human T cells. I. 20. Levin, S., M. Schlesinger, Z. Handzel, T. Hahn, Y. Altman, B. Czernobilsky, and Production of anti-Tac monoclonal antibody and distribution of Tac (+) cells. J. J. Boss. 1979. Thymic deficiency in Down’s syndrome. Pediatrics 63: 80–87. Immunol. 126: 1393–1397. 21. Murphy, M., and L. B. Epstein. 1990. Down syndrome (trisomy 21) thymuses 35. Bains, I., R. Antia, R. Callard, and A. J. Yates. 2009. Quantifying the de- have a decreased proportion of cells expressing high levels of TCR alpha, beta velopment of the peripheral naive CD4+ T-cell pool in humans. Blood 113: and CD3: a possible mechanism for diminished T cell function in Down syn- 5480–5487. drome. Clin. Immunol. Immunopathol. 55: 453–467. 36. Hazenberg, M. D., S. A. Otto, A. M. van Rossum, H. J. Scherpbier, R. de Groot, 22. Musiani, P., S. Valitutti, F. Castellino, L. M. Larocca, N. Maggiano, and T. W. Kuijpers, J. M. Lange, D. Hamann, R. J. de Boer, J. A. Borghans, and M. Piantelli. 1990. Intrathymic deficient expansion of T cell precursors in Down F. Miedema. 2004. Establishment of the CD4+ T-cell pool in healthy children syndrome. Am. J. Med. Genet. Suppl. 7: 219–224. and untreated children infected with HIV-1. Blood 104: 3513–3519. 23. Soares, M. V., N. J. Borthwick, M. K. Maini, G. Janossy, M. Salmon, and 37. Vrisekoop, N., R. van Gent, A. B. de Boer, S. A. Otto, J. C. Borleffs, A. N. Akbar. 1998. IL-7-dependent extrathymic expansion of CD45RA+ T cells R. Steingrover, J. M. Prins, T. W. Kuijpers, T. F. Wolfs, S. P. Geelen, et al. 2008. enables preservation of a naive repertoire. J. Immunol. 161: 5909–5917. Restoration of the CD4 T cell compartment after long-term highly active anti- 24. Demeure, C. E., D. G. Byun, L. P. Yang, N. Vezzio, and G. Delespesse. 1996. retroviral therapy without phenotypical signs of accelerated immunological ag- CD31 (PECAM-1) is a differentiation antigen lost during human CD4 T-cell ing. J. Immunol. 181: 1573–1581. maturation into Th1 or Th2 effector cells. Immunology 88: 110–115. 38. Albuquerque, A. S., C. S. Cortesaõ, R. B. Foxall, R. S. Soares, R. M. Victorino, and 25. Ernst, B., D. S. Lee, J. M. Chang, J. Sprent, and C. D. Surh. 1999. The peptide A. E. Sousa. 2007. Rate of increase in circulating IL-7 and loss of IL-7Ralpha ex- Downloaded from ligands mediating positive selection in the thymus control T cell survival and pression differ in HIV-1 and HIV-2 infections: two lymphopenic diseases with homeostatic proliferation in the periphery. Immunity 11: 173–181. similar hyperimmune activation but distinct outcomes. J. Immunol. 178: 3252–3259. 26. Haines, C. J., T. D. Giffon, L. S. Lu, X. Lu, M. Tessier-Lavigne, D. T. Ross, and 39. Bolotin, E., G. Annett, R. Parkman, and K. Weinberg. 1999. Serum levels of IL-7 D. B. Lewis. 2009. Human CD4+ T cell recent thymic emigrants are identified in bone marrow transplant recipients: relationship to clinical characteristics by protein tyrosine kinase 7 and have reduced immune function. J. Exp. Med. and lymphocyte count. Bone Marrow Transplant. 23: 783–788. 206: 275–285. 40. Fry, T. J., E. Connick, J. Falloon, M. M. Lederman, D. J. Liewehr, J. Spritzler, 27. Azevedo, R. I., M. V. Soares, J. T. Barata, R. Tendeiro, A. Serra-Caetano, S. M. Steinberg, L. V. Wood, R. Yarchoan, J. Zuckerman, et al. 2001. A potential

R. M. Victorino, and A. E. Sousa. 2009. IL-7 sustains CD31 expression in human role for interleukin-7 in T-cell homeostasis. Blood 97: 2983–2990. http://www.jimmunol.org/ naive CD4+ T cells and preferentially expands the CD31+ subset in a PI3K- 41. Franceschi, C., F. Licastro, P. Paolucci, M. Masi, S. Cavicchi, and M. Zannotti. dependent manner. Blood 113: 2999–3007. 1978. T and B lymphocyte subpopulations in Down’s syndrome: a study on non- 28. Prada, N., M. Nasi, L. Troiano, E. Roat, M. Pinti, E. Nemes, E. Lugli, institutionalised subjects. J. Ment. Deﬁc. Res. 22: 179–191. R. Ferraresi, L. Ciacci, D. Bertoni, et al. 2005. Direct analysis of thymic function 42. Dorshkind, K., E. Montecino-Rodriguez, and R. A. Signer. 2009. The ageing in children with Down’s syndrome. Immun. Ageing 2: 4. immune system: is it ever too old to become young again? Nat. Rev. Immunol. 9: 29. Hazenberg, M. D., S. A. Otto, J. W. Cohen Stuart, M. C. Verschuren, 57–62. J. C. Borleffs, C. A. Boucher, R. A. Coutinho, J. M. Lange, T. F. Rinke de Wit, 43. Akbar, A. N., and J. M. Fletcher. 2005. Memory T cell homeostasis and se- A. Tsegaye, et al. 2000. Increased cell division but not thymic dysfunction nescence during aging. Curr. Opin. Immunol. 17: 480–485. rapidly affects the T-cell receptor excision circle content of the naive T cell 44. Czesnikiewicz-Guzik, M., W. W. Lee, D. Cui, Y. Hiruma, D. L. Lamar, population in HIV-1 infection. Nat. Med. 6: 1036–1042. Z. Z. Yang, J. G. Ouslander, C. M. Weyand, and J. J. Goronzy. 2008. T cell 30. Comans-Bitter, W. M., R. de Groot, R. van den Beemd, H. J. Neijens, W. C. Hop, subset-speciﬁc susceptibility to aging. Clin. Immunol. 127: 107–118.

K. Groeneveld, H. Hooijkaas, and J. J. van Dongen. 1997. Immunophenotyping 45. Hu, H., M. R. Queiro`, M. G. Tilanus, R. A. de Weger, and H. J. Schuurman. by guest on September 27, 2021 of blood lymphocytes in childhood: reference values for lymphocyte sub- 1993. Expression of T-cell receptor alpha and beta variable genes in normal and populations. J. Pediatr. 130: 388–393. malignant human T cells. Br. J. Haematol. 84: 39–48. 31. de Vries, E., S. de Bruin-Versteeg, W. M. Comans-Bitter, R. de Groot, 46. Hu, H. Z., R. A. de Weger, K. Bosboom-Kalsbeek, M. G. Tilanus, J. Rozing, and W. C. Hop, G. J. Boerma, F. K. Lotgering, and J. J. van Dongen. 2000. Lon- H. J. Schuurman. 1992. T cell receptor V beta variable gene family expression in gitudinal survey of lymphocyte subpopulations in the ﬁrst year of life. Pediatr. human peripheral blood lymphocytes at the mRNA and membrane protein level. Res. 47: 528–537. Clin. Exp. Immunol. 88: 335–340.